In a living cell, such as a neuron, from the moment a primary RNA transcript is complete to the actual expression of the protein encoded by the transcript, multiple cellular events and mechanisms occur, including pre-RNA splicing, RNA editing, shuttling of the mRNA between the nucleus and the cytoplasm, and processes that ensure the stability and translational control of the trafficked mRNAs. Each of these events provides opportunities for the cell to regulate gene expression at the RNA level.
Each neuron is comprised of a nucleus within a body, or soma, a long fiber called the axon, and a varying number of branching fibers called dendrites, which extend out to other neurons. A single neuron can make numerous contacts with other neurons and tissues. For example, every new thought process is handled by a new set of synaptic connections. Memory itself is a set of synaptic connections engraved in the network of neurons.
Dendrites are specialized extensions of the neuronal soma that contain components of the cellular machinery involved in RNA and protein metabolism. A subset of mRNAs are trafficked to dendrites through their association with RNA binding proteins (RBPs). Some of these RBPs function in the nucleus as mediators of pre-RNA splicing.
The functional properties of neurites, including dendrites and axons, have been extensively examined since the discovery of protein synthetic machinery in dendrites, including ribosomes and membranous constituents of the endoplasmic reticulum and golgi apparatus (Bodian, 1965, Proc. Natl. Acad. Sci. U.S.A., 53:418-425; Steward et al., 1983, Res. 58:131-6; Torre et al., 1996, J. Neurosci. 16:5967-78; Gardiol et al., 1999, J. Neurosci. 19:168-79). Increasingly, more detailed molecular analyses of dendrites have shown that a subset of cellular RNAs are transported into dendrites where they can be translated into protein at specialized areas following synaptic stimulation (Aakalu et al., 2001, Neuron 30:489-502; Bassell et al., 1998, J. Neurosci. 18:251-65; Crino et al., 1996, Neuron 17:1173-87; Huber et al., 2000, Science 288:1254-7; Job et al., 2001, Proc. Natl. Acad. Sci. U.S.A 98:13037-42; Martin et al., 1997, Cell, 91:927-38). In the cytoplasm, the intracellular transport, stability, and translation of RNA are regulated by RNA binding proteins (RBPs) (Spirin et al., 1979, Mol. Biol. Rep. 5:53-57). RBP-RNA interactions typically occur through conserved motifs in RBPs that associate with cis acting sequences or secondary structures in RNA.
Recently, other RBPs thought to function only in the nucleus have also been localized in the cytoplasm. These include RNA editing enzymes (e.g. double stranded RNA adenosine deaminase) (Strehblow et al., 2002, Mol. Biol. Cell, 13:3822-35) as well as some of the highly conserved constituents of the spiceosome (e.g. the survival of motor neuron proton (Fan et al., 2002, Hum. Mol. Genet. 11:1605-14) and a variety of heterogeneous nuclear ribonucleoproteins (hnRNPs) (Pinol-Roma, 1997, Semin. Cell Dev. Biol. 8:57-63). Some auxiliary components of the spliceosome, such as the splicing factor SAM68, are present within the somatodendritric compartment of neurons as well (Staley et al., 1998, Cell 92:315-26; Jurica et al., 2003, Mol. Cell 12:5-14; Grange et al., 2004, J. Neurosci. Res., 75:654-66). The presence of these proteins in a non-nuclear compartment suggests that they either serve a unique functional role outside of the nucleus or their known functional activity can occur within this subcellular compartment.
The spliceosome, which catalyses the ATP-dependent removal of introns from nuclear pre-RNA, is a multi-megadalton complex of proteins and small nuclear RNAs (snRNA) (Staley et al., 1998, Cell 92:315-26; Jurica et al., 2003, Mol. Cell 12:5-14). Even in the nucleus, the distribution of pre-RNA splicing factors is not uniform. Rather, within discrete sites of concentration and lower levels of factors diffusely dispersed throughout the nucleoplasm, speckles (splicing factor compartments) can be readily identified with an antibody against the spliceosome assembly factor SC-35 (Lamond et al., 2003, Nat. Rev. Mol. Cell Biol. 4:605-12).
Despite the existing knowledge of these nuclear factors in the cytoplasm, the current state of the art does not definitively attribute function or role to the presence of RBPs and spliceosome components in the cytoplasm. A greater understanding of the regulation, metabolism and growth of cells will enable more accurate and more useful control and manipulation of cells. The development of such tools can enable more precise, targeted therapies and treatments of all mammals, and in particular, of humans. Therefore, there exists a need for a better understanding of the function and role of RBPs and spliceosome components in the cytoplasm in order to facilitate the controlled manipulation of cells. The present invention addresses and meets these needs.